Fibronectin (FN) is an integral part of virtually all tissues and an important link in cell recognition of environmental signals. FN functions as a major structural and functional component of the extracellular matrix, a structure that is indispensable for normal cell behavior. The structural diversity generated by alternative splicing may reflect functional differences required for FN's role in cell migration and adhesion, blood clotting, wound healing, and oncogenic transformation. The major goals of this proposal are to understand the molecular events that occur as FN is assembled into fibrils at the cell surface, to identify the regions that control cell adhesion and migration, and to localize elements that provide tissue-specific regulation of FN gene expression. We have developed a system to study matrix assembly based on the AtT2O cell line that does not express either FN or the alpha5beta1 FN receptor. AtT20+alpha5 cells engineered to express the receptor can assemble fibrils from FN added to the culture medium. Purified recombinant FNs (recFNs), expressed using mammalian and baculovirus expression systems, will be added and their assembly will be monitored by immunofluorescence microscopy and quantitated using labeled proteins. To study cell adhesion and migration, recFNs will be incorporated into fibrin-FN clots in vitro using our established techniques. Cells readily attach, spread, and migrate on the three-dimensional fibrin-FN matrix. Attachment and movement will be measured using optical sectioning by confocal microscopy and using cell specific markers. Another important modulator of FN function is variation in levels of gene expression. Sequences from upstream of the FN gene and from within introns will be analyzed for their ability to modulate transcription of the CAT reporter gene. Multiple enhancer and repressor sites have already been identified and will be further characterized. Elements involved in regulation during cell differentiation will also be mapped by using myoblasts as they fuse to form myotubes and embryonic stem cells which can differentiate along a number of cell lineages in vitro. Finally, recFNs and FN upstream regions will be combined to obtain tissue-specific expression in more complex, multi- component systems. This cohesive framework for characterizing the mechanisms and regulation of extracellular matrix assembly and function will provide the information needed to comprehend some very diverse biological processes.